This work was supported by NSF/DBI-1255991 grant to IML.

1. Preparação do capilar microrreactor <ol><li> Cortar o diâmetro interno 100 um (ID) x 360 uM exterior capilar diâmetro (DO) a um comprimento de 7-8 cm, e a 20 uM ID x 90 um OD capilar de 3-5 cm com o cutelo de capilar em vidro; verificar sob o microscópio que ambas as extremidades capilares têm um corte limpo, em linha reta, sem quaisquer rebarbas salientes. </li><li> Inserir a 20 um x 90 um ID OD capilar para um final de 100 uM ID x 360 uM OD capilar, para um comprimento de ~ 6 mm; em caso de necessidade, observar esta operação sob um microscópio. </li><li> Aplique uma pequena gota de E6000 cola em torno da junção dos 90 mm OD / 100 capilares mm ID com o fim de um Q-tip, e deixe a cola cura O / N à temperatura ambiente. </li><li> Cortar a 20 um x 90 um ID OD capilar inserido com um comprimento de ~ 10-15 mm; este vai ser o emissor de ionização por electropulverização. </li><li> Pré-cortar a 1/32 &quot;OD PEEK, 1/16&quot; PEEK OD e 1/16 &quot;banheira de PTFE ODing em pedaços de 4-5 cm de comprimento; estas serão as mangas utilizados em uniões capilares para fornecer uma conexão livre de vazamento. </li><li> Ligar a extremidade oposta da 100 mm Dl x 360 uM OD capilar para uma união PEEK; usar uma luva 1/32 &quot;PEEK para fornecer uma conexão livre de vazamento. </li><li> Inserir / apertar uma peça de ~ 5 cm de comprimento tubagem de PTFE 1/16 &quot;para a extremidade oposta da união. </li><li> Pesar ~ 4 mg de C18 (5 uM), em partículas de um pré-limpos / secos frasco de 2 ml de vidro; adicionar 0,5 ml de isopropanol, fechar o recipiente e dispersar a pasta no banho ultrasonicator. </li><li> Retirar com uma seringa de 250 ul a cerca de 200 pasta ul, inserir a agulha da seringa no &quot;tubo de 1/16 PTFE da união PEEK, e dispensar lentamente a pasta para o 100 mm ID x 360 mm OD capilar; observar ao microscópio como a capilar enche de partículas até um comprimento de 2-3 mm de embalagem é obtida, o que irá ser o micro-reactor (Figura 1) a 20 μ.m ID capilar retém as partículas na micro-reactor por meio de um efeito de distorção. 15 </li><li> Lavar o microrreactor com ~ 50 ul de solução de H2O / CH3CN 50:50 v / v, e em seguida, com solução de ~ 50 ul de H2O / CH3CN 98: 2 v / v. </li></ol> 2. Preparação das Soluções Amostra Nota: As operações que envolvam o manuseamento de solventes e ácidos orgânicos e preparação da solução são para ser executadas numa hotte. Usar óculos, luvas e vestuário de protecção. <ol><li> Lavar com CH3OH e secar alguns frascos de vidro (4 mL) e tubos de polipropileno (15 ml). </li><li> Preparar 10 ml de uma solução de H2O / CH3CN (98: 2 v / v) em um tubo de 15 ml de polipropileno por mistura de 9,8 ml de H2O com 200 ul de CH3CN; misture com ultra-sons. </li><li> Preparar 10 ml de uma solução de H2O / CH3CN (50:50 v / v) em um tubo de 15 ml de polipropileno por mistura de 5 ml de H 2 &lt;/ sub&gt; O com 5 ml CH 3 CN; misture com ultra-sons. </li><li> Prepare a 4 ml de uma solução aquosa acidificada (TFA a 0,01% v / v) através da adição de 4 mL de TFA (10%) a 4 ml de H 2 O / CH 3 CN (98: 2 v / v) em um frasco de vidro de 4 mL; misture com ultra-sons. </li><li> Prepare a 4 ml de uma solução orgânica acidificada (TFA a 0,01% v / v) através da adição de 4 mL de TFA (10%) a 4 ml de H 2 O / CH 3 CN (50:50 v / v) em um frasco de vidro de 4 mL; misture com ultra-sons. </li><li> Pesar 16 mg de NH 4 HCO 3 com uma microbalança analítica em um frasco de vidro de 4 mL; adicionar 4 ml de H 2 O / CH 3 CN: solução (98 2 v / v) e dispersa-se por ultra-sons; isto resulta em uma solução 50 mM de NH 4 HCO 3 (pH ~ 7,8) em tampão aquoso. </li><li> Pesar 5 mg de uma proteína padrão (por exemplo, albumina de soro bovino, a hemoglobina α / β, citocromo C, anidrase carbónica, α-2-HS-glicoproteína, etc) com uma microbalança analítica em um frasco de vidro de 4 mL; adicione 4 ml wate DIR e dispersar por sonicação; Isto normalmente resulta numa solução de elevada concentração de nível uM. </li><li> Prepare 1 ml de solução de proteína (1-2? M), diluindo a solução de concentração elevada de nível um, com um volume adequado de NH 4 HCO 3 (50 mM), tampão aquoso; misture com ultra-sons. </li><li> Preparar a solução de tripsina (5 ^ M) por dissolução de 20 ug de tripsina de grau de sequenciação-em 170 mL de NH 4 HCO 3 (50 mM), tampão aquoso; dispersar por sonicação. </li></ol> 3. Configuração Experimental Nota: O sistema LTQ-MS está equipado com uma fonte ESI modificado que inclui uma XYZ-estágio home-construído, que permite a interface do espectrômetro de massa para várias abordagens de entrada da amostra. <ol><li> Desligue o microreator capilar a partir da união PEEK e se conectar à PEEK Tee. </li><li> Insira fio Pt A ~ 2 cm de comprimento no lado-braço do PEEK T; utilizar uma &quot;manga PEEK 1/32 para fornecer umaconexão e para o isolamento do fio Pt livre de vazamento. </li><li> Conectar um 50 mm ID x 360 uM OD (~ 0,5 m de comprimento) de transferência das amostras capilar para a extremidade oposta do PEEK T; usar uma luva 1/32 &quot;PEEK para fornecer uma conexão livre de vazamento. </li><li> Fixe o Tee PEEK na XYZ-stage e posicione o ESI emissor ~ 2 mm de distância da massa capilar de entrada espectrômetro. </li><li> Ligue o fio de Pt à fonte de alimentação de energia ESI do espectrômetro de massa. </li><li> Ligar a extremidade oposta do capilar de transferência de amostras para a união de aço inoxidável; usar uma luva 1/16 &quot;PEEK para fornecer uma conexão livre de vazamento. </li><li> Conectar um 4-5 cm de comprimento &quot;1/16 tubagem de PTFE para a extremidade oposta da união de aço inoxidável e inserir a agulha da seringa de 250 ul na tubagem de PTFE; ligar a bomba e ajustar a taxa de fluxo no valor desejado (por exemplo, 2 ul / min). </li><li> em tandem entrada parâmetros MS de aquisição de dados para o pacote de software que controla o instrumento de MSd salvar como um arquivo método para pronta a configuração para realizar a análise; para o sistema LTQ-MS, use os parâmetros de análise dependentes seguintes dados: a exclusão dinâmica habilitado para 180 segundos com largura de massa exclusão ± 1,5 m / z; uma MS varredura seguido por um zoom e MS 2 varreduras em 5 melhores picos mais intensos, aumentar a largura de digitalização ± 5 m / z; dissociação induzida por colisão (CID) parâmetros definidos no isolamento largura de 3 m / z, energia de colisão normalizada de 35%, a activação Q 0.25, e tempo de activação 30 ms. </li></ol> 4. Setup Microfluidic <ol><li> Preparar um dispositivo de microfluidos de vidro ou substratos poliméricos; 16-18 a disposição do dispositivo é fornecida na Figura 2A, que consiste de um micro-reactor (1) (0,13 mm Comprimento largura x 2 mm x 50 mm de profundidade) ligada a uma entrada (2 ), saída (3) e uma porta lateral (4); os canais comunicantes para manipulações são fluídicos ~110 uM de largura e ~ 50 mm de profundidade; uma estrutura de múltiplos canais (5), que consiste em ~ 1,5-2 uM de profundidade x 10 m de largura x 100 mm de comprimento colocados microcanais de 25 um para além, irá actuar como um filtro para a retenção das partículas no microrreactor. </li><li> Ative as conexões de fluido de entrega para o chip por meio de colagem conectores especializados para as portas de entrada, ou através da elaboração de um suporte polimérico que acomoda acessórios para fornecimento de fluido (Figura 2B). 16 </li><li> Prepara-se uma pasta fluida de partículas de C18 (5 uM) tal como descrito no passo 1.8; fornecer a pasta para o micro-reactor com uma seringa usando 1/16 &quot;tubagem de PTFE ligado à porta lateral do chip (4); selar a porta após o carregamento de partícula com cola epóxi e cura durante 1 hora num forno a 90 ° C 16. </li><li> Inserir um capilar (20 uM ID x 90 um comprimento OD x 10 mm) no orifício de saída, selar com cola E6000, e deixar cura S / N; isso vai se tornar o emissor ESI ( <strong&gt; 6). </li><li> Conectar um 50 mm ID x 360 uM OD (~ 0,5 m de comprimento) de transferência das amostras capilar para o orifício de entrada do chip (2); ligar a extremidade oposta do tubo capilar a uma seringa de 250 ul e a bomba de seringa, tal como descrito em 3,6-3,7. </li><li> Coloque um leve conector PEEK T na linha de transferência de amostras imediatamente antes da abertura de entrada (2) e inserir um fio de Pt sobre o lado do braço do T, como descrito no ponto 3.2; este será o eletrodo ESI. </li><li> Fixar o dispositivo de microfluidos na fase XYZ com o emissor ESI posicionado ~ 2 mm de distância do capilar massa espectrómetro de entrada; preparar a MS para análise tal como descrito em 3.8. </li></ol> 5. Amostra de carregar, digestão proteolítica e eluição por análise por MS Nota: Todos os passos de transferência de solução / amostra são realizadas com o auxílio de uma bomba de seringa e deveria permitir a alguns minutos adicionais para completar, para compensar os mortos-volumes associados com as linhas de transferência de e para o micro-reactor; o tempo necessário dependerá das taxas de fluxo envolvidos. <ol><li> Lavar o microrreactor e prepará-la para a análise por infusão de uma solução aquosa de NH 4 HCO 3 (50 mM) em H 2 O / CH 3 CN (98: 2 v / v) em 2 ul / min durante 5 min; usar uma seringa de 250 ul. </li><li> Carregar a amostra de proteína (1 uM) no micro-reactor por infusão da solução de amostra a 2 mL / min durante 5 min; usar uma seringa de 250 ul. </li><li> Infundir a solução de tripsina (5 uM) sobre a proteína adsorvida no micro reactor em 2 ul / min durante 1-3 min. </li><li> Extingue-se o processo de digestão proteolítica, por lavagem do micro-reactor com uma solução aquosa acidificada de TFA (0,01% v / v) em H 2 O / CH 3 CN (98: 2 v / v) em 2 ul / min durante 5 min; usar uma seringa de 250 ul. </li><li> Iniciar eluindo a digestão de proteínas a partir do micro-reactor por infusão de uma solução orgânica acidificada de TFA (0,01%v / v) em H2O / CH3CN (50:50 v / v) em 300 nl / min. </li><li> Ligue o processo de aquisição de dados de MS e a voltagem ESI (~ 2000 V); adquirir dados de MS, utilizando os parâmetros de aquisição de dados fornecidos no passo 3.8; observar a eluição dos péptidos trípticos. </li></ol> 6. Processamento de Dados <ol><li> Processar os dados em tandem MS com um motor de busca compatível com o formato de dados MS-primas. <ol><li> Processar os arquivos raw LTQ-MS, utilizando um organismo banco de dados específico de proteína minimamente redundantes; fazer o upload dos dados brutos no motor de busca, definir as tolerâncias de massa pai e de iões fragmento a 2 e 1 Da, respectivamente, e usar apenas fragmentos totalmente trípticos com até dois perderam clivagens para a busca; não permitem modificações pós-translacionais; definir os de média e alta confiança falsos taxas de detecção (FDR) para 1% e 3%, respectivamente, e não permitem modificações pós-translacionais. </li></ol></li><li> Filtrar os dados para selecionar apenas de alta confiançapéptido corresponde às proteínas na base de dados; avaliar os resultados de proteína e de nível de péptido. </li></ol>

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The authors declare no competing financial interests.

O microreator descrito neste trabalho fornece um fácil de implementar configuração experimental para a realização de digestão enzimática de proteínas para permitir a análise MS e identificação em menos de 30 min. As vantagens deste sistema, em comparação com métodos convencionais, incluem a simplicidade, a velocidade e baixo consumo de reagentes e baixo custo. Em particular, não há necessidade de caros esferas de tripsina imobilizada e os cartuchos. A preparação do microreator capilar é simples <stron...

A identificação e caracterização das proteínas purificadas é frequentemente conseguido através de técnicas de MS. A proteína é digerido com uma enzima e os seus péptidos são ainda analisados ​​por MS utilizando uma configuração experimental simples de infusão. A digestão proteolítica é necessário para a geração de fragmentos peptídicos pequenos que caem na gama de massa da maioria dos analisadores útil MS, e que pode ser facilmente fragmentado por baixo de energia de colisão de dissociação induzida para gerar informação sobre a sequência de aminoácidos. Para as proteínas isoladas ou misturas de proteínas simples, não há mais necessidade de separação cromatográfica dos péptidos antes da detecção MS. Uma mistura de péptidos 25-50 podem ser facilmente analisadas por infusão da amostra com uma bomba de seringa directamente na fonte de iões de MS. O espectrómetro de massa pode realizar a análise e confirmar a sequência de uma proteína dentro de um curto espaço de tempo. Com os métodos modernos de aquisição de dados, este processo pode ser realizado Within alguns minutos ou mesmo segundos. O fator limitante para completar todo o processo em uma escala de tempo curta é a etapa de digestão proteolítica. Normalmente, isto é realizado ao longo de algumas horas (ou O / N), em solução, a 37 ºC, utilizando substrato: rácios de enzimas de (50-100): 1. Para reduzir o tempo de digestão enzimática para minutos ou segundos, microrreactores enzima imobilizada, sob a forma de reactores de microfluidos ou cartuchos disponíveis comercialmente, foram descritos. 06/01 Tipicamente, a enzima é imobilizada por ligação covalente, não-covalente / adsorção física, complexo formação ou encapsulamento, 3,6 a eficiência melhorada do processo enzimático a ser activada pela grande superfície para volume e rácios de enzima e substrato. As vantagens adicionais de reactores imobilizadas incluem a redução da autólise e interferência do enzima na análise de MS, melhorou a estabilidade da enzima e reutilização. Uma variedade de abordagens, utilizando vidro ou dispositivos microfabricados poliméricos têm sido descritos,usando enzimas imobilizadas em esferas magnéticas por interações antígeno-anticorpo, 7,8 aprisionados em redes de nanopartículas de ouro, 9 encapsulado em titânia de alumina sol-gel 10 e nanozeolites, 11 ou capturados através de Ni-NTA ou His-Tag formação do complexo. 6 Alternativamente , capilares tubulares abertas com enzimas imobilizadas têm sido desenvolvidos, assim. 12 além disso, a clivagem proteolítica aumentada foi demonstrada utilizando a irradiação de microondas controlado 13 ou assistida por pressão ou ciclagem tecnologia pressão (PCT) para reduzir os tempos de reacção a 30-120 min. 14 Apesar das várias vantagens de reactores de enzimas imobilizadas, os custos de cartuchos comerciais é alta, a disponibilidade de dispositivos de microfluidos para uso de rotina é limitado, e o uso de tecnologias de micro-ondas ou de PCT resultados em necessidade de instrumentação adicional. O objetivo deste trabalho foi desenvolver um método que circumvents estas desvantagens, e que pode ser facilmente implementado em cada laboratório para capacitar investigadores com uma abordagem simples e eficaz para a realização de clivagem enzimática das proteínas na preparação para análise por MS em poucos minutos. A abordagem baseia-se na utilização de, C18-partículas hidrofóbicas, que são pré-carregados em um capilar ou dispositivo de microfluidos, e a adsorção da proteína (s) de interesse sobre essas partículas, seguido por digestão enzimática durante a infusão da enzima sobre o de leito fixo e de proteína (s) capturado. Nesta abordagem, o substrato é imobilizada através de interacções não covalentes, e a enzima é infundido através da proteína imobilizada. A eficiência proteolítica digestão é aumentada pelas grandes áreas de superfície de partícula que expõem a proteína para o processamento enzimático, as distâncias e os tempos de difusão de e para a superfície de partículas reduzido, uma melhor transferência de massa, sem ligação covalente que pode afectar a actividade da enzima, a capacidade Avaliado com a rapideze combinações de diferentes enzimas, descartável, e a multiplexação, se o processo é executado num formato de microfluidos. Esta abordagem é demonstrada com o uso de uma mistura de proteínas padrão e tripsina a enzima mais comumente utilizado para a digestão proteolítica antes da detecção ESI-MS. O espectrómetro de massa utilizados para a detecção neste estudo foi um instrumento linear armadilha de quadrupolo (LTQ).

<table><tbody><tr><td>Ion trap ESI-MS</td><td>Thermo Electron</td><td>LTQ</td><td>The LTQ mass spectrometer is used for acquiring tandem MS data</td></tr><tr><td>XYZ stage</td><td>Newport</td><td>Multiple parts</td><td>The home-built XYZ stage is used to adapt the commercial LTQ nano-ESI source to receive input from various sample delivery systems</td></tr><tr><td>Stereo microscope</td><td>Edmund optics</td><td>G81-278</td><td>The microscope is used to observe the microreactor packing process</td></tr><tr><td>Analytical balance/Metler</td><td>VWR</td><td>46600-204</td><td>The balance is used to weigh the protein samples</td></tr><tr><td>Ultrasonic bath/Branson</td><td>VWR</td><td>33995-540</td><td>The sonic bath is used for mixing/homogenizing the samples and dispersing the C18 particle slurry</td></tr><tr><td>Syringe pump 22</td><td>Harvard Apparatus</td><td>552222</td><td>The micropump is used for loading, rinsing and eluting the sample and the enzyme on and from the packed capillary microreactor</td></tr><tr><td>Milli-Q ultrapure water system</td><td>EMD Millipore</td><td>ZD5311595&amp;nbsp;</td><td>The MilliQ water system is used to prepare purified DI water</td></tr><tr><td>Pipettor/Eppendorf (1,000&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-410</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (100&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-406</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (10&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-402</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Fused silica capillary (100 &amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP100375</td><td>This capillary is used for the fabrication of the microreactor</td></tr><tr><td>Fused silica capillary (20&amp;nbsp;&amp;micro;m ID x 100 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP020090</td><td>This capillary is used for the fabrication of the ESI emitter</td></tr><tr><td>Fused silica capillary (50&amp;nbsp;&amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP050375</td><td>This capillary is used to transfer the samples and the eluent from the syringe pump to the capillary microreactor</td></tr><tr><td>Glass capillary cleaver</td><td>Supelco</td><td>23740-U</td><td>This is a tool for cutting fused silica capillaries at the desired length</td></tr><tr><td>Glue</td><td>Eclectic Products</td><td>E6000 Craft</td><td>This glue is used for securing the ESI emitter into the capillary microreactor or the microfluidic chip</td></tr><tr><td>Epoxy glue</td><td>Epo-Tek</td><td>353NDT</td><td>This glue is used to seal the microfluidic inlet hole through which the C18 particles are loaded</td></tr><tr><td>Reversed phase C18 particles (5&amp;nbsp;&amp;micro;m)</td><td>Agilent Technologies</td><td>Zorbax 300SB-C18</td><td>These are C18 particles on which the proteins are adsorbed; the particles were extracted from a 4&amp;nbsp;mm x 20 cm C18 LC column from Agilent</td></tr><tr><td>Syringe/glass (250 &amp;micro;l)</td><td>Hamilton</td><td>81130-1725RN</td><td>The glass syringes are used to load the C18 particle slurry in the capillary microreactor and to deliver the sample and eluents to the microreactor</td></tr><tr><td>Internal reducing PEEK Union (1/16&amp;rdquo; to 1/32&amp;rdquo;)</td><td>Valco</td><td>ZRU1.5FPK</td><td>This union is used to connect the 250 &amp;micro;l syringe to the microreactor for loading the 5 &amp;micro;m particle slurry</td></tr><tr><td>Stainless steel union (1/16&amp;rdquo;)</td><td>Valco</td><td>ZU1XC</td><td>The stainless steel union is used to connect the glass syringe needle to the infusion capillary</td></tr><tr><td>Microvolume PEEK Tee connector (1/32&amp;rdquo;)</td><td>Valco</td><td>MT.5XCPK</td><td>The Peek tee is used to connect the sample transfer capillary to the capillary microreactor; on its side arm, it enables the insertion of the Pt wire</td></tr><tr><td>Tee connector (light weight)</td><td>Valco</td><td>C-NTXFPK</td><td>This Tee connector is used to apply ESI voltage to the microfluidic chip through the sample transfer line</td></tr><tr><td>Pt wire (0.404 mm)</td><td>VWR</td><td>66260-126</td><td>The Pt wire provides electrical connection for ESI generation and is connected to the mass spectrometer ESI power supply</td></tr><tr><td>PTFE tubing (1/16&amp;rdquo; OD)</td><td>Valco</td><td>TTF115-10FT</td><td>The Teflon tubing is used to enable an air-tight connection between the syringe needle and the stainless steel union&amp;nbsp;</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/16&amp;rdquo; OD)</td><td>Upchurch Scientific</td><td>1565</td><td>The Peek tubing is used as a sleeve to enable an air-tight connection between the stainless steel union and the 50&amp;nbsp;&amp;micro;m ID transfer capillary</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/32&amp;rdquo; OD)</td><td>Valco</td><td>TPK.515-25</td><td>The Peek tubing is used as a sleeve to enable a leak-free connection between the fused silica capillaries and the Peek Tee</td></tr><tr><td>Clean-cut polymer tubing cutter</td><td>Valco</td><td>JR-797</td><td>This cutter is used to pre-cut the 1/16&amp;rdquo; and 1/32&amp;rsquo; Peek polymer tubing that is used as sleeve for leak-free connections in pieces of ~4-5 cm in length</td></tr><tr><td>Amber vial (2 ml)</td><td>Agilent&amp;nbsp;</td><td>HP-5183-2069</td><td>The vials are used to prepare sample solutions and the C18 particle slurry&amp;nbsp;</td></tr><tr><td>Amber vial (4 ml)</td><td>VWR</td><td>66011-948</td><td>The vials are used to prepare sample solutions</td></tr><tr><td>Polypropylene tube (15&amp;nbsp;ml)</td><td>Fisher</td><td>12-565-286D</td><td>The vials are used to prepare buffer solutions</td></tr><tr><td>Cylinder (100 ml)</td><td>VWR</td><td>24710-463</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Cylinder (10 ml)</td><td>VWR</td><td>24710-441</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Pipette tips (1,000 &amp;micro;l)</td><td>VWR</td><td>83007-386</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (100 &amp;micro;l)</td><td>VWR</td><td>53503-781</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (10 &amp;micro;l)</td><td>VWR</td><td>53511-681</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Glass substrates</td><td>Nanofilm</td><td>B270 white crown, 3&amp;rdquo; x 3&amp;rdquo;</td><td>These are glass substrates for microchip fabrication</td></tr><tr><td>Male nut fitting (1/16&amp;rdquo;)</td><td>Upchurch</td><td>P203X</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Nanoport assembly</td><td>Upchurch</td><td>N-122H</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Protein standards</td><td>Sigma</td><td>Multiple #</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher</td><td>A955</td><td></td></tr><tr><td>Methanol, HPLC grade</td><td>Fisher</td><td>A452</td><td></td></tr><tr><td>Isopropanol, HPLC grade</td><td>Sigma</td><td>650447</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma</td><td>302031</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Aldrich</td><td>A6141</td><td></td></tr><tr><td>Trypsin, sequencing grade</td><td>Promega</td><td>V5111</td><td></td></tr></tbody></table>

fast enzymatic processing of proteins for ms detection with a flow-through microreactor

A grande maioria de espectrometria de massa (MS) baseados em métodos de análise de proteínas envolvem um passo de digestão enzimática antes da detecção, tipicamente com tripsina. Este passo é necessário para a geração de péptidos de baixo peso molecular, geralmente com PM &lt;3000-4000 Da, que caem dentro do intervalo de varrimento efectivo de instrumentação de espectrometria de massa. protocolos convencionais envolvem O / N digestão enzimática a 37 ºC. Os avanços recentes levou ao desenvolvimento de uma variedade de estratégias, que envolvem normalmente a utilização de um micro-reactor com enzimas imobilizadas ou de uma variedade de processos físicos complementares que reduzem o tempo necessário para a digestão proteolítica de alguns minutos (por exemplo, microondas ou alta pressão). Neste trabalho, nós descrevemos um método simples e de baixo custo que pode ser implementado em qualquer laboratório para a realização rápida digestão enzimática de uma proteína. O (ou mistura de proteína) proteína é adsorvida em alta perf de fase inversa C18-ligadoormance partículas de sílica com cromatograf ia líquida (HPLC), pré-carregados numa coluna capilar, e com tripsina em tampão aquoso é administrada por perfusão durante as partículas para um curto período de tempo. Para permitir a detecção em linha de MS, os peptídeos trípticos são eluídas com um sistema de solvente com o aumento do teor orgânico directamente na fonte de iões de MS. Esta abordagem evita a utilização de partículas de alto preço enzima imobilizada e não necessitam de qualquer ajuda para completar o processo. a digestão de proteínas e a análise da amostra completa pode ser realizada em menos de 3 minutos ~ e ~ 30 min, respectivamente.

Um resultado representativo de um processo de digestão proteolítica realizado simultaneamente com uma mistura de proteínas, com os micro-reactores acima descritos (Figuras 1 ou 2), é fornecida na Tabela 1. A tabela compreende as sequências de péptidos únicas que identificam uma proteína em particular, a cruz o resultado de correlação (Xcorr) (isto é, uma pontuação que caracteriza a qualidade do jogo experimental-a-teórica para o es...

Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Enzimática processamento rápido de proteínas para a detecção MS com um micro-reator de fluxo-through

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Research

JoVE Journal

Bioengineering

7.9K visualizações.  Virginia Tech. A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

Vídeo: Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

resolvida no tempo com ionização por electrospray Hidrogénio-deutério troca Espectrometria de Massa para estudar a estrutura e dinâmica das proteínas

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Bioquímica

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

Monitoramento em tempo real de Reações realizada utilizando processamento de fluxo contínuo: A Preparação de 3-Acetylcoumarin como Exemplo

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Química

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Um Método de Extração Fracionada Simples para a Análise Abrangente de Metabolitos, Lípidos e Proteínas de uma única amostra

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Misturadores microfluídicos para Estudar o dobramento de proteínas

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Bioengenharia

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Uma preparação de amostra de plasma para espectrometria de massa usando uma estação de trabalho automatizada

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

A new static platform is used to characterize protein structure and interaction sites in the native cell environment utilizing a protein footprinting technique called in-cell fast photochemical oxidation of proteins (IC-FPOP).

Incubadora de plataformas com estágio XY móvel: uma nova plataforma para implementar oxidação fotoquímica rápida em células de proteínas

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

A microfluídica digital para processamento automatizado Proteômica

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biologia

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Analisar grandes complexos de proteínas por espectrometria de massa estruturais

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Proteômica por Espectrometria de Massas Guiada por Linhagem Celular no Embrião em Desenvolvimento (Rã)

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...